2480 Journal of Applied Microscopy 



Schaffer, J. Versuche mit Entkalkungsflus- The author has made an exhaustive 

 sigkeiten. Zeitsch. f. wiss. Mik. 19 : 308-328, series of experiments to determine the 

 44 - 3' 9 3- most satisfactory reagent for decalci- 



fying vertebrate tissues and the most successful method of applying it. He has 

 tested practically all of the acids recommended for decalcifying in various 

 strengths, and in some cases in combination with alcohol or phloroglucin. He 

 has also determined the best method of removing the acid from the preparations. 

 The most complete decalcification with a minimum of deformation due to shrink- 

 age, swelling, or dissolution of the tissue, was obtained by carefully embedding 

 well-fixed tissues in celloidin, hardening the celloidin block in 85 per cent, alco- 

 hol, grading down through lower per cents, of alcohol to water, exposure for 12 

 to 24 hours (longer for large masses of tissue) to 3 to 5 per cent, (by weight) 

 aqueous solution of nitric acid in a Thoma's water wheel. From the acid the 

 block is passed to a 5 per cent, solution of lithium or sodium sulphate for 12 to 

 24 hours. This solution should be changed at least once. The sulphate is 

 removed by washing for 48 hours in running water, after which the object is 

 again graded up to 85 per cent, alcohol. 



Phosphoric, lactic, formic, and acetic acids, even in concentrated solutions, 

 were found to produce swelling in tissues containing collagen, and their dissolv- 

 ing power is also so slight as to preclude their use in decalcification. Formic 

 or acetic acid may, however, be used in connection with reagents which prevent 

 swelling, or on tissues fixed in osmic acid or formalin, but never on fresh tissues 

 or on those fixed by ordinary methods. 



The author finds that the following acids are available for use on heavily 

 calcified tissues, such as bones and teeth ; viz., hydrochloric, nitric, trichlora- 

 cetic, and sulphuric, named in order of their decalcifying capacity. Swelling is 

 caused by the trichloracetic and still more by the sulphuric acid, increasing after 

 washing out the acid with the first-named, but diminishing after the last. Both pro- 

 duce precipitates in the tissues, which are, however, easily soluble in water and 

 therefore necessitate careful washing out after decalcification. This second group 

 of acids produce no swelling in tissues fixed in formalin when they are used in 

 solutions of 3 to 10 per cent. Long action of the hydrochloric acid afiiects the 

 chromatin and reduces its stainability. For this reason nitric acid is to be pre- 

 ferred to the hydrochloric. Its dissolving power is but a little less than that of 

 the hydrochloric acid, and it leaves the tissues, cells and nuclei in much finer 

 condition, and retains their elective coloration. 



The nitric acid may be used without damage to tissues in strengths of 2 to 

 10 per cent., but since 10 per cent, or even 20 per cent, decalcifies no more 

 rapidly than 5 per cent, it is unnecessary to exceed this strength. This 5 per 

 cent, aqueous solution (by weight) will decalcify without trace of swelling 42 

 grams of solid bone in 10 hours. Additions to the nitric acid tend to prolong 

 the decalcification, phloroglucin but slightly, formalin still more, and alcohol most 

 of all. Low grades of alcohol not only tend to prolong the decalcification but 

 also swell and shorten the fibrillar. Five per cent, alum solution does not affect 

 the rapidity of action of the acid, but otherwise furthers the decalcification. All 

 these additions to the acid have been employed to prevent swelling, but the 2 to 



